Tumor necrosis factor (TNF) is a cytokine produced mainly by activated macrophages which elicits a wide range of biological effects. These include an important role in endotoxic shock and in inflammatory, immunoregulatory, proliferative, cytotoxic, and anti-viral activities (reviewed by Goeddel, D. V. et al., Cold Spring Harbor Symposia on Quantitative Biology 51, 597-609 [1986]; Beutler, B. and Cerami, A., Ann. Rev. Biochem. 57, 505-518 [1988]; Old, L. J., Sci. Am. 258(5), 59-75 [1988]; Fiers, W. FEBS Lett. 285(2), 199-212 [1991]). The induction of the various cellula responses mediated by TNF is initiated by its interaction with two distinct cell surface receptors, an approximately 55 kDa receptortermed TNF-R1 and an approximately 75 kDa receptor termed TNF-R2. Human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher, H. et al., Cell 61, 351 [1990]; Schall, T. J. et al., Cell 61, 361 [1990]; Smith, C. A. et al., Science 248, 1019 [1990]; Lewis, M. et al., Proc. Natl. Acad. Sci. USA 88, 2830-2834 [1991]; Goodwin, R. G. et al., Mol. Cell. Biol. 11, 3020-3026 [1991]. Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular portions of both receptors are found naturally also as soluble TNF-binding proteins (Nophar, Y. et al., EMBO J. 9, 3269 [1990] and Kohno, T. et al., Proc. Natl. Acad. Sci. U.S.A. 87, 8331 [1990]). The amino acid sequence of human TNF-R1 and the underlying nucleotide sequence are disclosed in EP 417,563 (published Mar. 20, 1991), whereas EP 418,014 (published Mar. 20, 1991) discloses the amino acid and nucleotide sequences of human TNF-R2.
Both TNF receptors are independently active in signaling TNF responses. Direct signaling by TNF-R2 has been observed in lymphoid cells in which TNF-R2 stimulates the proliferation of thymocytes and a murine cytotoxic T cell line CT6 (Tartaglia et al., Proc. Natl. Acad. Sci. USA 88, 9292-9296 [1991]; Tartaglia et al, J. Immunol. 151, 4637-4641 [1993]). Both TNF-R1 and TNF-R2, along with other members of the TNF receptor superfamily, e.g. CD40, have been shown to independently mediate the activation of the transcription factor NF-.kappa.B (Lenardo & Baltimore, Cell 58: 227-229 [1989]; Laegreid, A., et al., J. Biol. Chem. 269, 7785-7791 [1994]; Rothe et al., Cell 78, 681-692 [1994]; Wiegmann et al., J. Biol. Chem. 267, 17997-18001 [1992]). NF-.kappa.B is a member of the Rel family of transcriptional activators that control the expression of a variety of important cellular and viral genes (Lenardo & Baltimore, supra, and Thanos and Maniatis, Cell 80, 529-532 [1995]). TNF-R2 also mediates the transcriptional induction of the granulocytemacrophage colony stimulating factor (GM-CSF) gene (Miyatake et al., EMBO J. 4: 2561-2568 [1985]; Stanley et al., EMBO J. 4: 2569-2573 [1985]) and the A20 zinc fingerprotein gene (Opipari et al., J. Biol. Chem. 265: 14705-14708 [1990]) in CT6 cells, and participates as an accessory component to TNF-R1 in the signaling of responses primarily mediated by TNF-R1, like cytotoxicity (Tartaglia, L. A. and Goeddel, D. V., Immunol. Today 13, 151-153 [1992]).
Recent research has lead to the isolation of polypeptide factors associated with the intracellulardomain of the 75 kDa tumor necrosis factor receptor, TNF-R2 ("tumor necrosis factor receptor associated factors" or "TRAFs") which participate in the TNF-R2 signal transduction cascade. TRAF1 and TRAF2 were the first two identified members of this novel protein family containing a novel C-terminal homology region, the TRAF domain (Rothe et al., Cell 78, 681-692 [1994]; U.S. Pat. Nos. 5,670,319 issued on Sep. 23, 1997, and 5,708,142 issued Jan. 13, 1998. A further TRAF domain protein, TRAF3 (originally termed CD40bp, CRAF, or LAP1) has also been identified (Hu et al., J. Biol. Chem. 269, 30069 [1994]; Cheng et al., Science 267, 1494 [1995], and Mosialos et al., Cell 80, 389 [1995]). TRAFs transduce signals from TNF-R2, CD40 and presumably from other members of the TNF receptor superfamily that also includes the low affinity nerve growth factor receptor, the Fas antigen, CD27, CD30, OX40, 4-1BB, and TNFR-RP (Rothe et al., supra; Hu et al., J. Biol. Chem. 269, 30069-30072 [1994]; Cheng et al., Science 267, 1494-1498 [1995]; Mosialos et al., Cell 80, 389-399 [1995]; Rothe et al., Science, in press; Smith et al, Cell 76, 959-962 [1994]). In addition to the shared conserved C-terminal TRAF domain that is involved in both receptor association and oligomerization, TRAF2 and TRAF3 each contain an N-terminal RING finger domain and five zinc finger structures of weak sequence similarity. CD40 and TNF-R2 interact directly with TRAF2 and indirectly with TRAF1 via a TRAF2/TRAF1 heterodimer. TRAF2 (or the TRAF2/TRAF1 heterodimer) is required for CD40- and TNF-R2-mediated activation of the transcription factor NF-.kappa.B. TRAF3 interacts with CD40 and self-associates, but does appear to not associate with TNF-R2, TRAF1, or TRAF2. The role of TRAF3 in signal transduction is less well defined, but it may antagonize the effects of TRAF2 (Rothe et al., Science, in press). The TRAF proteins also interact with the C-terminal cytoplasmic domain of the Epstein-Barr virus transforming protein LMP1 (Mosialos et al., Cell 80, 389 [1995]). LMP1 is a dominant oncogene that has multiple downstream effects on cell growth and gene expression, at least some of which require NF-.kappa.B activation (Laherty et al., J. Biol. Chem. 267, 24157 [1992]; Rowe et al., J. Virol. 68, 5602 [1994]). TRAF2 is believed to be a common signal transducer for TNF-R2, CD40 and LMP1.